GENERATION OF FUNCTIONAL HUMAN DENDRITIC CELLS FROM ADHERENT PERIPHERAL-BLOOD MONOCYTES BY CD40 LIGATION IN THE ABSENCE OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR

Recently it has been shown that dendritic cells (DC) can develop from peripheral blood monocytes when grown in the presence of granulocyte-m acrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). However, it is unclear whether DC can also develop from monocytes in absence of these cytokines. We therefore analyzed the effect of Flt-3 ligand (Flt3L) and of CD40 ligand on the development of human DC from blood monocytes in the absence of GM-CSF Adherent peripheral blood mon onuclear cells (PBMNC) were cultured in the presence of different cyto kine combinations and analyzed for the expression of surface molecules and antigen presenting capacity. For functional analyses, cells were tested for their ability to stimulate allogeneic T lymphocytes in a mi xed lymphocyte reaction (MLR), to present soluble antigens, and to ind uce primary HIV-peptide-specific cytotoxic T-cell (CTL) responses in v itro. Furthermore, expression of DC-CK1, a recently identified chemoki ne with specific expression in DC, and of IL-18 (IGIF), a growth and d ifferentiation factor for Th 1 lymphocytes, was analyzed by reverse-tr anscription polymerase chain reaction (RT-PCR). In our study, Flt3L al one was not sufficient to generate DC and required addition of IL-4. D C generated with Flt3L and IL-4 underwent maturation after stimulation with tumor necrosis factor-alpha (TNF-alpha) or CD40L, characterized by CD83 expression, upregulation of MHC, adhesion, and costimulatory m olecules as well as increased allogeneic proliferative response. In co ntrast, CD40 ligation alone promoted differentiation of adherent blood monocytes into functional DC in the absence of GM-CSF and IL-4. These cells displayed all phenotypic and functional characteristics of matu re DC and were potent stimulatory cells in priming of major histocompa tibility complex (MHC) class I-restricted CTL responses against an HIV -peptide, whereas their ability to present soluble protein antigens wa s reduced. Using a semiquantitative RT-PCR, DC-CK1 and IL-18 transcrip ts were detected in all generated DC populations, independent of growt h factors used. Our findings provide further evidence for the importan ce of CD40-CD40L interaction for initiation and maintenance of T-cell responses and confirm the emerging concept that blood monocytes provid e an additional source of DC depending on external stimuli. (C) 1998 b y The American Society of Hematology.